Viability and activity in readily culturable bacteria: a review and discussion of the practical issues.

In microbiology the terms 'viability' and 'culturability' are often equated. However, in recent years the apparently self-contradictory expression 'viable-but-nonculturable' ('VBNC') has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms 'viability' and 'culturability' in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces 'VBNC' with expressions that are internally consistent.

Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP.

The general stress-induced sigma subunit sigma s of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor sigma 70. In view of their very similar promoter specificity in vitro, it is unclear how sigma factor selectivity in the expression of sigma s-dependent genes is generated in vivo. The csiD gene is such a strongly sigma s-dependent gene. In contrast to sigma s, which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only. To our knowledge, the csiD promoter is the first characterized promoter which is not only exclusively dependent on sigma s-containing RNA polymerase (E sigma s), but also requires an activator, cAMP-CRP. In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression. Also in vitro, E sigma s is more efficient than E sigma 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD -35 region to the sigma 70 consensus and to transcription by E sigma s being less dependent on contacts in this region. By DNase I protection experiments, a cAMP-CRP binding site centered at -68.5 nucleotides upstream of the csiD transcriptional start site was identified. While cAMP-CRP stimulates E sigma 70 binding, it does not promote open complex formation by E sigma 70, but does so in conjunction with E sigma s. With linear templates, cAMP-CRP significantly stimulates E sigma s-mediated in vitro transcription, whereas transcription by E sigma 70 is negligible and hardly stimulated by cAMP-CRP. These findings may reflect different or less stringent positional requirements for an activator site for E sigma s than for E sigma 70, and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter. In vitro transcription experiments with super-coiled templates, however, revealed significant cAMP-CRP-stimulated transcription also by E sigma 70. Yet, under these conditions, H-NS was found to restore E sigma s specificity by strongly interfering with cAMP-CRP/E sigma 70-dependent transcription. Lrp strongly and cooperatively binds to multiple sites located between positions -14 and -102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with E sigma s. In summary, we conclude that the csiD promoter has an intrinsic preference for E sigma s, but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong E sigma s selectivity. Furthermore, this strong E sigma s preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP ensures csiD expression under conditions of carbon starvation, but not other stress conditions.

In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.

Plasmid Transfer between Marine Vibrio Strains during Predation by the Heterotrophic Microflagellate Cafeteria roenbergensis.

The effect of grazing by the heterotrophic microflagellate Cafeteria roenbergensis on plasmid transfer between marine Vibrio S14 strains was studied by using artificial seawater. Several factors of potential importance for regulation of the plasmid transfer, such as nutrient release, production of a flagellate-derived substance(s) that may affect plasmid transfer, and the presence of surfaces, were investigated. Only living flagellates gave rise to, at instances, plasmid transfer enhanced more than 2 orders of magnitude. We propose that the activity of grazing flagellates allows for the significant increase in plasmid transfer observed under predation conditions. This may be due to a localized increase in bacterial numbers through filter feeding, thus providing high cell densities with increased possibility for cell-to-cell-contact and hence plasmid transfer.

Analysis of starvation conditions that allow for prolonged culturability of Vibrio vulnificus at low temperature.

The response of the estuarine human pathogen Vibrio vulnificus to starvation for carbon, nitrogen or phosphorus, or all three nutrients simultaneously (multiple-nutrient), was examined with respect to the maintenance of culturability during incubation at low temperature. V. vulnificus showed similar survival patterns during starvation for the individual nutrients when kept at 24 degrees C. On the other hand, cultures prestarved at 24 degrees C and then shifted to 5 degrees C maintained culturability at low temperature in a starvation-condition-dependent manner. Carbon and multiple-nutrient starvation were indistinguishable in their ability to mediate maintenance of culturability in the cold. Prolonged starvation for phosphorus had a similar effect, but nitrogen starvation did not allow for maintenance of culturability. Extracellular factors produced during starvation were not observed to have an effect on the culturability of cells incubated at low temperature. Protein synthesis during starvation for individual nutrients was analysed by two-dimensional PAGE of pulse-labelled proteins. Carbon and multiple-nutrient starvation gave nearly identical protein induction patterns involving at least 34 proteins, indicating that carbon starvation determines both responses. Nitrogen starvation for 1 h induced 24 proteins, while phosphorus starvation induced a set of 10 proteins after 1 h and about 40 proteins after 18 h. It is suggested that starvation for carbon or phosphorus induces maintenance of culturability of V. vulnificus incubated at low temperature via the synthesis of distinct sets of starvation-specific proteins.

Identification and characterization of stationary phase-inducible genes in Escherichia coli.

During transition into stationary phase a large set of proteins is induced in Escherichia coli. Only a minority of the corresponding genes has been identified so far. Using the lambda placMu system and a plate screen for carbon starvation-induced fusion activity, a series of chromosomal lacZ fusions (csi::lacZ) was isolated. In complex medium these fusions were induced either during late exponential phase or during entry into stationary phase. csi::lacZ expression in minimal media in response to starvation for carbon, nitrogen and phosphate sources and the roles of global regulators such as the alternative sigma factor sigma s (encoded by rpoS), cAMP/CRP and the relA gene product were investigated. The results show that almost every fusion exhibits its own characteristic pattern of expression, suggesting a complex control of stationary phase-inducible genes that involves various combinations of regulatory mechanisms for different genes. All fusions were mapped to the E. coli chromosome. Using fine mapping by Southern hybridization, cloning, sequencing and/or phenotypic analysis, csi-5, csi-17, and csi-18 could be localized in osmY (encoding a periplasmic protein), glpD (aerobic glycerol-3-phosphate dehydrogenase) and glgA (glycogen synthase), respectively. The other fusions seem to specify novel genes now designated csiA through to csiF. csi-17(glpD)::lacZ was shown to produce its own glucose-starvation induction, thus illustrating the intricacies of gene-fusion technology when applied to the study of gene regulation.

How do non-differentiating bacteria adapt to starvation?

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.

Low temperature induced non-culturability and killing of Vibrio vulnificus.

Vibrio vulnificus cells progressively lose culturability during incubation at 5 degrees C. This process is accelerated by the addition of supernatants from non-culturable cells obtained by incubation at 5 degrees C for 17 days. Thus the organism apparently produces a factor upon cold incubation which is triggering or causing the decline in culturability. Reversing the temperature shift can restore a culturable population comparable in numbers to the original population, but this process is largely due to regrowth. A few cells retaining the ability to grow apparently utilize the substrates released by the moribund cells, thus mimicking resuscitation of the whole population.